P593: T-CELL DYSFUNCTION BY PSEUDOHYPOXIA AND AUTOCRINE PURINERGIC SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA
Authors: Chiara Montironi, Chaja F Jacobs, Gaspard Cretenet, Fleur Peters, Arnon Kater, Helga Simon-Molas, Eric Eldering
Journal: HemaSphere (2023)

Background: Acquired T-cell dysfunction is a prominent feature in chronic B-cell malignancies, such as chronic lymphocytic leukemia (CLL). This is widely assumed to hinder the efficacy of T-cell based immunotherapy. T cells rely on metabolic switches to initiate and sustain an immune response, namely a rapid shift to glycolysis accompanied by amplification of OXPHOS capacity. Aims: Given the link between T-cell metabolism and function, we aimed at uncovering T-cell metabolic alterations induced by malignant B cells as the basis of T-cell dysfunction. Methods: Using B-cell malignant cell lines and human PBMCs, we first established a model which recapitulates major aspects of cancer-induced T-cell dysfunction. PBMCs derived from healthy donors were co-cultured with malignant B-cell lines from different origin, namely PGA-1 (CLL), LME-1 (multiple myeloma) and Nalm-6 (acute lymphoblastic leukemia), and analyzed by flow cytometry, extracellular flux analyzer, transcriptomics and metabolomics. In second instance, we investigated whether primary CLL cells induce similar metabolic alterations in autologous T cells, using samples of CLL patients enrolled in the 1st-line clinical trial investigating MRD-driven Venetoclax and Obinutuzumab treatment (Ho139; Netherlands Trial Registry ID: NTR6043). Results: Exposure to the CLL-derived cell line PGA-1 during stimulation with anti-CD3/28, resulted in defective activation, proliferation and effector function of CD4 and CD8 T cells. Altered function of T cells upon co-culture required cell-cell contact. PGA-1 cells altered the T-cell metabolome by generating a pseudohypoxic state, characterized by the imbalance of the metabolites α-ketoglutarate and succinate which leads to HIF-1α stabilization in presence of oxygen. HIF-1α but not HIF-2α target genes were upregulated. Pseudohypoxia contributed to T-cell dysfunction by two mechanisms. Firstly, T cells were retained in aerobic glycolysis and not able to switch to OXPHOS, leading to reduced T-cell metabolic fitness. Moreover, pseudohypoxic T cells upregulated the enzymatic machinery to produce - and sense - adenosine in an autocrine manner. This negatively affected T-cell function by dampening activation, which could be restored by specific blocking of adenosine receptors A2AR/A2BR by the dual-antagonist AB928. We next uncovered a similar hypoxic-like signature in autologous T cells from primary CLL samples, also caused by imbalance of the metabolites α-ketoglutarate and succinate, lending clinical relevance to our findings. Pseudohypoxia was reversible upon depletion of CLL cells in vitro and, importantly, upon reduction of the leukemic burden after treatment with venetoclax and obinutuzumab, restoring T-cell function. Summary/Conclusion: In conclusion, we uncover a pseudohypoxic program connected with T-cell dysfunction in CLL. This novel mechanistic insight suggests that modulation of hypoxia and the purinergic pathway might contribute to therapeutic restoration of T-cell function. Keywords: B cell chronic lymphocytic leukemia, Cancer immunotherapy